1. EXPLANATION

Trehalose is a disaccharide that occurs naturally in insects, plants, fungi, and bacteria. The major dietary source is mushrooms. Trehalose is used in bakery goods, beverages, confectionery, fruit jam, breakfast cereals, rice, and noodles as a texturizer, stabilizer, humectant, or formulation aid with a low sweetening intensity. Trehalose has not previously been considered by the Committee.

2. BIOLOGICAL DATA

2.1 Biochemical aspects

2.1.1 Absorption and distribution

In general, the fate of ingested or parenterally administered trehalose corresponds to that of glucose since trehalose is rapidly hydrolysed to glucose by the enzyme trehalase. Trehalase is found in humans and most animals at the brush border of the intestinal mucosa, as well as in the kidney, liver, and blood plasma (Hore & Messer, 1968; van Handel, 1970; Demelier et al., 1975; Labat-Robert, 1982; Niederst & Dauça, 1985; Eze, 1989; Riby et al., 1990; Yoshida, 1993). Trehalase activity has been found in the small intestine of humans, mice, rats, guinea-pigs, rabbits, pigs, and baboons (Cerda et al., 1972; Hietanen, 1973; Ruppin et al., 1974; Maestracci, 1976; Garland, 1989). No trehalase activity is found in the small intestine of cats (Hore & Messer, 1968; Hietanen, 1973; Garland, 1989).

A very few individuals have trehalase deficiency, which may be hereditary or acquired. However, in Greenland, the prevalence of trehalase deficiency has been reported to be 8%, which is considerably higher than that seen elsewhere (Dahlqvist, 1974; Gudmand-Høyer et al., 1988). The incidence of trehalase deficiency is lower than that of lactase deficiency, which in the United Kingdom is 3.2–6% (Gudmand-Høyer & Skovbjerg, 1996). When trehalose is ingested by such individuals, it is either incompletely digested or undigested, and a small fraction (approximately 0.5%) may be absorbed by passive diffusion, as shown for other disaccharides (van Elburg et al., 1995). The absorbed trehalose may then be metabolized to glucose in the liver or kidney or be excreted unchanged in the urine (Demelier et al., 1975). Unabsorbed trehalose is likely to be fermented by the intestinal microflora to short-chain fatty acids such as acetate, propionate, and butyrate.

In a study of the relative absorption of trehalose and glucose as an indicator of malabsorption, 50 healthy volunteers were given 50 g of trehalose or 50 g of glucose on different days, and the blood glucose concentration was examined before treatment and 15, 20, 60, 90, and 120 min after treatment. The fraction of trehalose absorbed as glucose over the first 60 min was calculated from the ratio of the area under the curve of the concentration of glucose in blood with time after trehalose administration and that after glucose administration. For normal individuals, the observed ratio ranged from 0.3 to 1.5, with a median of 0.7 (Bergoz et al., 1973). An abnormal ratio (< 0.26) was associated with malabsorption resulting from a disease of the small intestine. In a similar study, conducted with nine patients with chronic renal failure, the absorption of trehalose relative to glucose was 0.83 (Pointner et al., 1974).

In a study of the correlation between oral administration of various sugars and exhalation of hydrogen, 60 healthy volunteers received 50 g of trehalose in 400 ml of water. Blood was collected for determination of glucose before treatment and 30, 60, 90, and 120 min after treatment. The reported absorption rate (median, 0.7) indicated that trehalose is incompletely digested to glucose in the small intestine (Heine et al., 1996).

2.1.2 Biotransformation and excretion

Trehalose entering the circulating blood is converted to glucose by trehalase in serum, kidney, liver, and bile, depending on the species (van Handel, 1969; Labat-Robert, 1982; Arola et al., 1999). The trehalose that is not converted to glucose is excreted in the urine of rats, guinea-pigs, and birds, which lack renal trehalase. In mice, rabbits, dogs, pigs, and humans, trehalase in the brush border of the proximal tubular cells of the kidney would be expected to cleave the excreted trehalose to glucose (Demelier et al., 1975; Niederst & Dauça, 1985; Riby et al., 1990).

In a study of the metabolism of absorbed trehalose, groups of rats, guinea-pigs, and rabbits were given an intravenous dose of 0.5 or 1 g/kg bw. In rats, 87% of the administered dose was recovered from the urine, indicating that there is little or no trehalase activity in the liver and kidneys. In guinea-pigs and rabbits, only 7–9% of the administered dose was recovered from the urine. Examination of the trehalose concentration in the renal artery and vein of rabbits indicated that it was hydrolysed efficiently in the kidneys (Demelier et al., 1975).

In a study of renal trehalase activity, rabbits were given 500 mg of trehalose intravenously, corresponding to 200–300 mg/kg bw. Trehalose was cleared from the plasma within 60 min, and none was found in the urine. In contrast, in rats given 100 mg of trehalose intravenously (for a dose of about 300 mg/kg bw), trehalose was cleared from the plasma at the same rate as in rabbits but was found in the urine in proportion to the plasma concentration. This indicates that no renal metabolism of trehalose occurred in rats (Riby et al., 1990).

In a study of parenteral use of trehalose as a source of saccharide, groups of rabbits were given intravenous doses of trehalose (10% solution), maltose (10% solution), or glucose (5% solution) for 90 min at 6.7 ml/kg bw per h. Blood was collected before treatment and at 30, 60, 90,120, and 180 min. Infusion of trehalose led to a rapid increase in serum glucose concentration that returned to normal 90 min after cessation of infusion. Only about 1% of the infused trehalose was recovered in the urine (Sato et al., 1999).

2.2 Toxicological studies

2.2.1 Acute toxicity

The acute toxicity of trehalose was examined in mice, rats, and dogs. The results are summarized in Table 1, and further details given below.

Table 1. Acute toxicity of trehalose in male and female animals

Species

Route

LD50 (mg/kg bw)

Reference

Mouse

Oral

> 5 000

Atkinson & Thomas (1994a)

Mouse

Intravenous

> 1 000

Atkinson & Thomas (1994a)

Rat

Oral

> 16 000

McRae (1995)

Rat

Oral

> 5 000

Atkinson & Thomas (1994b)

Rat

Intravenous

> 1 000

Atkinson & Thomas (1994b)

Dog

Oral

> 5 000

Atkinson & Thomas (1994c)

Dog

Intravenous

> 1 000

Atkinson & Thomas (1994c)

Groups of five male and five female CD-1 mice and Sprague-Dawley rats received a single dose of trehalose at 1 g/kg bw by intravenous injection into the tail vein or 5 g/kg bw by oral gavage. A control group of each species received either intravenous or oral doses of sterile saline at the same volumes. Four male beagle dogs received a single dose of trehalose at 1 g/kg bw by intravenous injection into the cephalic vein. After a recovery period of 6 days, 5 g/kg bw were administered orally in capsules. All animals of all species were observed twice daily for signs of toxicity, and blood was taken at intervals to determine serum glucose concentrations. In the studies with mice and rats, satellite groups consisting of five animals of each sex per group were treated identically to the treated groups, and blood and urine were collected for analysis of glucose and trehalose.

Mice showed no signs of toxicity after administration by either route, nor were there any treatment-related changes in body weight during the 14-day observation period. Serum glucose concentrations rose slightly but significantly 30 min after oral administration of trehalose but were comparable thereafter. Trehalose was detected in plasma and urine after intravenous administration and in the urine only after oral dosing. The glucose concentration in urine was increased after administration by either route (Atkinson & Thomas, 1994a).

The rats showed no signs of toxicity after administration by either route, nor were there any treatment-related changes in body weight during the 14-day observation period. A slight increase in serum glucose concentration was seen during the first 2 h after oral dosing, but no changes in that in the plasma were seen. Trehalose was detected in plasma and urine after intravenous administration and in the urine only after oral dosing. The glucose concentration in the urine was increased after administration by either route (Atkinson & Thomas, 1994b).

In the dogs, no signs of toxicity were observed after administration by either route, nor were there any treatment-related changes in body weight during the 7-day observation period. Slight but transient changes in serum glucose concentration were seen after administration by either route. Trehalose was detected in the plasma after either route of administration but in the urine only after intravenous administration. The concentration of glucose in urine was increased after administration by either route (Atkinson & Thomas, 1994c).

In a separate study, five male and five female Sprague-Dawley rats were given trehalose orally at a dose of 16 g/kg bw. There were no deaths and no clinical signs of toxicity (McRae, 1995).

Trehalose was instilled at a volume of 0.1 ml of a 10% solution into the right eye of each of six New Zealand white rabbits. The left eye of each animal was used as a control, and each eye was scored for irritation 1, 24, 48, and 72 h after application. The study was terminated at day 4. There were no deaths or signs of systemic toxicity and no evidence of irritation to the cornea, iris, or conjunctivae. Under the conditions of this assay, trehalose was not an eye irritant (Atkinson & Thomas, 1994d).

2.2.2 Short-term studies of toxicity

Mice

In a 14-day study, groups of 10 CD-1 mice of each sex were given trehalose (purity not stated) orally at a dose of 5 g/kg bw per day by gavage, subcutaneously at a dose of 2.5 mg/kg bw per day, or intravenously into the tail vein at a dose of 1 g/kg bw per day. The animals were observed throughout the study, and body weights were recorded and blood collected at intervals for haematological and clinical chemical evaluations. The mice were killed on day 15, and various tissues were preserved for histological examination.

One animal died on day 6 after intravenous dosing, but there had been no clinical signs on previous days, and the findings at necropsy were unremarkable. There were no treatment-related clinical signs of toxicity or treatment-related effects on body weights. Food consumption was normal in all groups. The serum glucose concentration rose slightly after intravenous or subcutaneous treatment but quickly returned to normal. This effect was considered to be transient, and treatment did not lead to accumulation of glucose. The leukocyte counts of males were reduced 14 days after oral or subcutaneous dosing, and this was attributed to a decrease in total lymphocytes. A similar decrease occurred after intravenous dosing but was not statistically significant. There were no accompanying histopathological changes in the bone marrow and no haematological changes in females in any group. Slight increases in sodium and potassium concentrations were seen in males treated by all three routes. The phosphorus concentration was also increased in males after subcutaneous dosing. Males dosed orally had decreased blood urea nitrogen and increased albumin and total protein. Macroscopic changes were observed only in animals treated intravenously, and these were restricted the tail. Histological examination of tissues revealed no remarkable findings (Atkinson & Thomas, 1994e).

Groups of 20 HanIbm.NMRL mice of each sex were fed a pelleted maintenance diet to which trehalose (purity, 99.2%) was added at a concentration of 0, 5000, 15 000, or 50 000 mg/kg (equal to 0, 760, 2200, or 7300 mg/kg bw per day for males and 0, 910, 2700, or 9300 mg/kg bw per day for females) for 13 weeks. The mice were observed daily for clinical signs of toxicity, body weights were measured weekly, and ophthalmoscopic examinations were performed before treatment and at week 13 on control and high-dose animals. Blood and urine samples were collected at 5, 9, and 13 weeks for clinical biochemistry, haematology, and urinary analysis. At 13 weeks, the animals were necropsied, and a range of tissues and organs was collected. Tissue samples from control and high-dose animals and from animals that died during the study were examined histologically.

Two animals in the control group, one at the low dose, and one at the high dose died during treatment, but these deaths were considered not to be related to treatment. There were no treatment-related signs of toxicity and no treatment-related ophthalmoscopic changes. The body weights of male mice at the two higher doses were slightly reduced throughout the study period, but those of mice at the highest dose were significantly reduced only at week 12. The body weights of females were similar in all groups. Haematological parameters were unaffected by treatment, and only sporadic changes were observed. A slight increase in plasma glucose concentration was seen in males and females at the high dose at weeks 5, 9, and 13. In females, this difference was statistically significant at weeks 5 and 9. Animals at the two lower doses also showed a slight increase in blood glucose concentration at weeks 5 and 9, but the difference was statistically significant only in females at the intermediate dose at week 9. The plasma bilirubin concentration was significantly reduced in males and females at the highest dose during week 5 and in males only in week 9. The plasma calcium concentration was significantly decreased in males and females at the two higher doses at week 13 only. The plasma phosphorus concentration was slightly but significantly increased in males and females at the highest dose at week 5 and in females at the two higher doses at week 9. A slight but nonsignificant increase in plasma phosphorus concentration was observed in both males and females at week 13; however, a treatment-related effect on plasma phosphorus concentration seems unlikely. The plasma potassium concentration could be measured only at week 13 (because of insufficient blood), and a statistically significant decrease was observed in males at the two higher doses and in females at the highest dose. No treatment-related effects were found on urinary parameters.

There were no treatment-related effects on organ weights or appearance. Histological examination of a wide range of tissues revealed no treatment-related effects. While sporadic changes in clinical parameters were observed in this study, they did not show a consistent, treatment-related pattern. The NOEL was 50 000 mg/kg of diet, equal to 7300 mg/kg bw per day, the highest dose tested (Schmid et al., 1998).

Dogs

In a 14-day study, groups of three beagles of each sex were given trehalose (purity not stated) orally at 5 g/kg bw per day by capsule, subcutaneously at 0.25 g/kg bw per day, or intravenously at 1 g/kg bw per day into the cephalic vein. The control group received empty capsules daily for 14 days. The animals were checked daily for signs of toxicity and weighed weekly. Blood was collected for haematology and clinical chemistry.

No deaths occurred during the study, and the animals treated intravenously showed no signs of toxicity. All dogs treated orally had diarrhoea, and one control and two female animals regurgitated the capsule. One male treated subcutaneously had diarrhoea on one occasion. Body weights and body-weight changes were similar in treated and control animals. There was no treatment-related effect on food consumption. The serum glucose concentrations were normal in all groups, except for a slight increase during the first hour after intravenous treatment.

Haematological parameters were normal after oral or subcutaneous treatment. Males showed slight but significant decreases in erythrocyte count, and females had a decreased mean corpuscular volume and mean corpuscular haemoglobin concentration. No significant changes were observed at 14 days. A slight increase in blood urea nitrogen was seen in females treated orally, but as no changes were noted in males treated orally or in males or females treated subcutaneously the significance of this result is questionable. Sporadic macroscopic changes were observed in individual animals, but these were not related to treatment. The histological findings were unremarkable (Atkinson & Thomas, 1994f).

2.2.3 Genotoxicity

The results of tests for the genotoxicity of trehalose are summarized in Table 2.

Table 2. Results of tests for the genotoxicity of trehalose

End-point

Test object

Concentration

Result

Reference

Reverse mutation

S. typhimurium TA1535, TA1537, TA98, TA100; E. coli WP2 uvrA

310, 620, 1250, 2500, 5000 µg/platea

Negative

Kitching (1995)

Chromosomal aberration

Chinese hamster ovary cells

1250, 2500, 5000 µg/ml

a

Negative

Winegar (1997a)

Micronucleus formation

Male and female mice

1250, 2500, 5000 mg/kg bw

Negative

Winegar (1997b)

a

With and without microsomal activation

2.2.4 Reproductive toxicity

(a) Multigeneration studies

Rats

In a two-generation study, groups of 28 Wistar rats of each sex were fed a pelleted maintenance diet to which trehalose (purity, 99%) was added at a concentration of 0, 25 000, 50 000, or 100 000 mg/kg, equivalent to doses of 0, 1.25, 2.5, and 5 g/kg bw per day. Animals of the F0 generation were mated after 10 weeks on the diet to produce the F1 generation and were kept on the modified diet until sacrifice after weaning of the F1 animals. On post-natal day 4, the litters were culled to groups of four animals of each sex per dose, and on post-natal day 21 the pups were weaned and groups of 28 of each sex were chosen randomly to rear the next (F2) generation. Animals of the F1 generation were treated in the same way as the F0 animals and were mated after 10 weeks to produce the F2 generation. All animals were observed for clinical signs, and reproductive and clinical parameters were examined in each generation.

No treatment-related clinical signs of toxicity were seen during pre-mating, gestation, or lactation in the F0 or F1 animals. Sporadic differences in body weights were seen between groups at various times, but with no dose-dependent relationship; the differences were considered not to be related to treatment. Similar sporadic differences were observed in food consumption but were also considered not to be related to treatment. Macroscopic examination of the parental F0 and F1 animals revealed no treatment-related changes, and histological examination of selected tissues revealed no remarkable difference. Fertility and reproductive parameters were normal in each generation, and there were 24, 26, 24, and 27 and 23, 25, 25, and 25 pregnant females in the control, low-, mid-, and high-dose groups of the F0 and F1 generations, respectively. There was no treatment-related effect on the fecundity index or the fertility index. The duration of gestation was comparable in all groups, as was the number of liveborn pups. The gestation index was 100% for all groups of both generations. Significant increases and decreases in litter size were seen between groups in both the F0 and F1 generations, but these differences were not dose-related and were not consistent across generations and were therefore considered not to be related to treatment. The sex ratio was comparable in all groups on postnatal days 1 and 21 in both generations. The numbers of small and large pups showed some differences between groups, but these differences were sporadic and considered unrelated to treatment. There were no grossly malformed pups and no abnormal findings at macroscopic examination. There were no treatment-related differences in body weight or body-weight gain between the control and treated groups of the F0 or F1 generation. The NOEL was 100 000 mg/kg of diet, equivalent to 5 g/kg bw per day, the highest dose tested (Wolterbeek & Waalkens-Berendsen, 1999a).

(b) Developmental toxicity

Rats

Groups of 28 mated female Wistar rats were fed diets containing trehalose (purity, 99%) at a concentration of 0, 25 000, 50 000, or 100 000 mg/kg, equal to mean dietary intakes of 0, 1.7, 3.5, and 6.9 g/kg bw per day, on days 0–21 days of gestation. The dams were examined throughout the study for clinical signs of toxicity, and their organs were examined macroscopically at the end of the study. On day 21, the dams were killed, and the fetuses from the control and high-dose groups were examined for visceral and skeletal abnormalities.

No deaths occurred during the study, and clinical examination revealed remarkable findings in only two animals, one in the control group and one at the high dose, which had a haemorrhagic discharge from the vagina on days 21 and14 of gestation, respectively. Examination of the organs did not reveal any difference in adverse changes between the treated and control groups. The mean body weights of the treated pregnant animals did not differ from those of the control animals. Food consumption was normal in all groups. No differences between treated and control groups were found in the numbers of corpora lutea, implantations, live and dead fetuses, and early and late resorptions. The rates of pre- and post-implantation loss and the sex ratios of the fetuses were also similar. There were no treatment-related effects on the weights of the reproductive organs weights or on maternal body weights during gestation.

Gross examination of the fetuses revealed a significantly decreased number of large fetuses in dams at the low and high doses and a significantly decreased number of small fetuses in those at the low dose. These differences were likely to be incidental and not related to treatment. One fetus at the low dose had a flexed hindlimb, and one fetus in each of the control and high-dose groups had a filiform tail. No other macroscopic lesions were observed. The mean fetal and placental weights were similar in treated and control animals. Visceral examination of the fetuses in the control and high-dose groups revealed no malformations, and there were no significant differences in the incidences of visceral anomalies and variations. Skeletal examination of the fetuses revealed no malformations in the control or high-dose groups. The incidence of skeletal anomalies and variations was unremarkabke, and there was no difference in the incidence in the control and high-dose groups. No differences were seen in skeletal ossification, except for a slight but significant difference in the incidence of ossification in the phalanges of the hindlimbs. In the absence of effects at other sites, this difference was considered not to be related to treatment. The NOEL was 100 000 mg/kg of diet, equal to 6.9 g/kg bw per day, the highest dose tested (Waalkens-Berendsen, 1998).

Rabbits

Groups of 16 mated New Zealand white rabbits were fed diets containing trehalose (purity, 99%) at at a concentration of 0, 25 000, 50 000, or 100 000 mg/kg, equal to intakes of 0, 0.77, 1.3, and 2.8 g/kg bw per day, on days 0–7 of gestation, which were reduced to intakes of 0, 0.21, 0.48, and 1.0 mg/kg bw per day on days 21–29 of gestation. On day 29 of gestation, the dams were killed and examined macroscopically, and the fetuses were examined for visceral and skeletal malformations.

There were no treatment-related signs of toxicity in the dams during treatment. Gross examination of the organs and tissues did not reveal any treatment-related changes, and no treatment-related differences in body weight were seen. The food consumption was normal in all groups, but the rate of intake of trehalose decreased in all groups during gestation. The fecundity index was 75, 75, 87, and 81% for the control, low-, mid-, and high-dose groups, respectfully. The numbers of corpora lutea, implantations, live and dead fetuses, early and late resorptions, and pre- and post-implantation losses and the sex ratio of the fetuses did not differ from controls. The organ weights and maternal body-weight changes during gestation were similar in all groups.

Examination of the fetuses revealed no significant differences between controls and treated groups with regard to external findings, placental weight, or fetal weight. Examination of the viscera of the fetuses showed no treatment-related malformations, anomalies, or variations. Examination of the skeleton showed one that fetus in the control group had fused ribs. The incidence of skeletal anomalies was similar between the groups. The only difference in skeletal variations was a significant increase in the incidence of accessory ribs in animals at the intermediate dose. No difference was seen in the high-dose group. Because of the lack of a dose–response relationship and because the difference was seen only on a litter basis, the variation is unlikely to be related to treatment. Incidental but significant differences in variations in skeletal ossification were seen, such as a decrease in the incidence of incompletely ossified thoracal bodies and an increase in the incidence of fetuses with unossified distal epiphyses of the humerus in fetuses at the intermediate dose, but these changes were considered not to be related to treatment. The NOEL was 100 000 mg/kg of diet, equal to 2.8 g/kg bw per day, the highest dose tested (Wolterbeek & Waalkens-Berendsen, 1999b).

Malto-oligosyl trehalose synthase (EC 5.4.99.15) and malto-oligosyl trehalose trehalohydrolase (EC 3.2.1.141) are obtained from Arthrobacter ramosus (Nakada et al., 1995a,b), which is a soil microorganism generally considered to be non-pathogenic. No toxicological studies were available on these enzymes; however, purification of trehalose results in almost complete removal of proteinaceous material.

(c) Isoamylase

Isoamylase (EC 3.2.1.68) is obtained from a mutant strain of Pseudomonas amyloderamosa. In a study of acute toxicity, groups of five male and five female mice received a 40% aqueous suspension of isoamylase at concentrations ranging from 12 000 to 21 000 mg/kg of diet. The LD50 was approximately 15 000 mg/kg of diet (Morimoto et al., 1979)

In a short-term study of toxicity, groups of 20 Wistar rats of each sex were given isoamylase by gavage at a dose of 0, 2.3, 5, or 10 ml/kg bw per day, equivalent to 0, 57, 110, or 230 mg of protein per kg bw per day, or 0, 4.1, 8.2, or 16 million units of enzyme activity per kg bw per day. The animals were examined throughout the study for clinical signs, and body weights were measured weekly. Blood was collected at the end of the study for haematology and blood chemistry, and urinary analysis was conducted. Ophthalmoscopic examinations were performed on control and high-dose animals at the beginning and end of the study. All animals were necropsied at 3 months, and selected tissues were taken for microscopic examination.

Five animals died during the study, but the deaths were considered not to be treatment-related, as there were no clinical signs of toxicity in the surviving animals. The results of the ophthalmoscopic examinations were normal. There were no treatment-related changes in mean body weight, and food and water consumption were normal in all groups. A significant increase in haemoglobin concentration was found in females at the low and high doses, but the absence of a dose–response relationship and of other haematological changes suggests that these changes were not treatment-related. There were no other differences between control and treated groups and no significant differences in clinical chemical or urinary parameters between the control and treated groups. At necropsy, no treatment-related changes in absolute or relative organ weights were found, and the only gross pathological changes seen were those associated with gavage. Histopathological examination revealed no changes associated with treatment. The NOEL was 10 ml/kg bw per day, equivalent to 230 mg of protein per kg bw per day (Lina, 1999).

Isoamylase was tested for its ability to induce reverse mutation at the his locus in Salmonella typhimurium strains TA1535, TA1537, TA98, and TA100, and at the trp locus in Escherichia coli WP2uvrA with and without metabolic activation, at concentrations of 62–5000 µg of protein per plate. Isoamylase did not cause a reproducible increase in the number of revertants of any of the bacterial strains, with or without metabolic activation (van Delft, 1999).

(d) Cyclodextrin glucanotransferase

Cyclodextrin glucanotransferase (EC 2.4.1.19) is obtained from a strain of Bacillus stearothermophilus. Data on the toxicity of cyclodextrin glucanotransferase from other source organisms were considered previously by the Committee in the context of its assessements of the safety of beta-cyclodextrin and gamma-cyclodextrin, and no concern was raised (Annex 1, references 107, 116, and 138). The safety of B. stearo-thermophilus as a source organism was considered previously in the context of the evaluation of the safety of alpha-amylase from this organism (Annex 1, reference 94), in which it was concluded that B. stearothermophilus is non-pathogenic to humans and animals.

(e) Glucoamylase and alpha-amylase from

Bacillus subtilis

Glucoamylase (EC 3.2.1.3) obtained from Aspergillus niger and alpha-amylase from Bacillus subtilis were evaluated previously by the Committee (Annex 1, references 77 and 94). An ADI of 0–1 mg of total organic solids per kg bw was established for glucoamylase (amylglucosidase) from A. niger, and an ADI ‘not specified’ was established for alpha-amylase from B. subtilis.

2.3 Observations in humans

2.3.1 Studies of tolerance in healthy volunteers

In a study of potential adverse effects, 60 healthy volunteers were given 50 g of trehalose in 400 ml of water after an overnight fast. No abdominal symptoms of diarrhoea were reported (Bolte et al., 1973).

In a study in which 10 volunteers were given a single dose of 25 g of trehalose in 200 ml of water 1 h after breakfast, there was no reported diarrhoea or other abdominal symptoms (Heine et al., 1996).

In an investigation of the laxative dose of trehalose, 20 female students were given a single daily dose of trehalose in 200 ml of solution 2–3 h after a meal. The dose of trehalose was increased gradually from 10 to 20, 30, 40, 50, and 60 g per treatment. The indigestible disaccharide lactulose was used as a positive control. The physical condition and gastrointestinal symptoms of all subjects were recorded before and after treatment. Half of the subjects had no gastrointestinal symptoms, even at the highest dose tested (60 g), whereas lactulose caused diarrhoea in 75% of subjects after ingestion of 40 g. Both trehalose and lactulose caused high prevalences of abdominal symptoms including flatulence, distension, and borborygmus, but the effects of lactulose were significantly more severe than those of trehalose at the same dose. While there was significant variation between individuals, the threshold dose for transitory laxation was estimated to be 0.65 g/kg bw for trehalose and 0.26 g/kg bw for lactulose (Oku & Okazaki, 1998).

In a study of the absorption of trehalose, 30 healthy adults (15 of each sex) were given trehalose at a single oral dose of 10, 20, 30, or 40 g. The concentrations of hydrogen gas in expired air and of glucose in blood were measured before and every 30 min after administration for 3 h. Subjects were considered to be suffering from malabsorption if the hydrogen gas concentration in expired air increased by more than 20 ppm from the standard value. The subjects were also examined for gastrointestinal symptoms (malabsorption, abdominalgia, laxation, abdominal dysphoria, and crepetus). The rates of malabsorption of trehalose were 0%, 40%, 43%, and 75% at 10, 20, 30, and 40 g, respectively, while the rates of gastrointestinal symptoms were 0%, 40%, 43%, and 50%. Malabsorption was found in more than half of the subjects given 40 g of trehalose. While no racial differences in the ability to absorb trehalose were seen between Mongoloid (Japanese), white, and black individuals, the Japanese had a significantly higher incidence of gastrointestinal symptoms (Ushijima et al., 1995).

2.3.2 Studies of trehalase-deficient individuals

Few cases of heriditary or acquired trehalase deficiency have been reported. The first was that of a women who noted that intake of mushrooms provoked diarrhoea before the end of the meal. A subsequent test for tolerance to trehalose confirmed her intolerance to this sugar, although this may not have been the only cause (Bergoz, 1971). A second case was confirmed by biopsy from the upper jejunum, which showed the absence of intestinal trehalase (Madzarovova-Nohejlova, 1973). Two further cases were reported by Bergoz et al. (1982).

In order to determine whether malabsorption of trehalose causes abdominal symptoms and to establish the most suitable diagnostic tools for distinguishing intolerant from tolerant subjects, a trehalose load test was performed on 64 subjects. The persons were asked to consume 25 g of trehalose after an overnight fast, and blood glucose concentration and hydrogen and methane in the breath were measured. Trehalase activity was measured in a biopsy sample from the duodenum. Of the 19 individuals who were intolerant to mushrooms, 13 experienced flatulence and abdominal distension and six experienced diarrhoea; however, relative trehalase deficiency was detected in only two subjects. The concentrations of gases in breath and of glucose in blood did not differ between the groups. Although the results suggest that symptoms are the best indicator of trehalase deficiency, the study did not include a placebo treatment to allow for over-reporting of intestinal side-effects (Arola et al., 1999).

The incidence of trehalase deficiency is unknown, owing to the limited amount of data, but it has been reported to be 2% (Bergoz et al., 1982). In other studies, however, no cases of intolerance were detected in 123 (Welsh et al., 1978) and 248 subjects (Gudmand-Høyer et al., 1988).

In a study to determine the normal range of trehalase activity in a population in the United Kingdom, duodenal biopsy samples were taken from 400 patients with suspected malabsorption for histological assessment and estimation of trehalase. In 369 patients with normal histological appearance, the distribution of trehalase activity was normal (4.8– 37 U/g protein). One patient had borderline trehalase deficiency. The 31 patients with villous atrophy had a diagnosis of coeliac disease and significantly reduced activity of disaccharidases, including trehalase. When these patients were placed on a gluten-free diet, the activities of maltase, sucrase, and trehalase returned to normal in most, whereas the lactase activity did not recover. The authors concluded that there is no basis for routine determination of trehalase activity in the population of the United Kingdom (Murray et al., 2000),

3. ESTIMATED DIETARY INTAKE

Trehalose has not been approved for use throughout the world, and its intake has not been estimated. However, predictions may be made on the assumption of maximum levels of use in named food categories in combination with reported data on food consumption. The intake of trehalose was predicted for the populations of Australia and the USA.

The intakes predicted in Australia were based on the maximum levels of use in nine food categories, with chewing-gum included as confectionery (Table 3) and on data on food consumption derived from a national nutrition survey of the whole population aged 2 years and over in 1995, comprising 13 858 individual dietary records based on a single 24-h recall. The range of predicted intakes, on the assumption that trehalose is present at the lower and upper limits of the range of concentration, were 5.7–9.7 g/day for all respondents and 6.5–11 g/day for 12 258 consumers only (Australia–New Zealand Food Authority, 2000).

Table 3. Food categories and concentrations reported by the manufacturer and data on food consumption for the whole population (aged ł 2 years) of Australia

Food category

Concentration
of trehalose (%)

Mean food consumption by all respondents (g/day)

3.1.1

Ice cream

10

16

4.3.4

Fruit and vegetable spreads

10–20

4.8

5

Confectionery

7–20

10

5.4

Icings and frostings

5

0.1

6.1

Whole and broken grains (rice)

2

19

6.4

Flour products (including pasta)

2

14

7.2

Biscuits, cakes, and pastries

5–10

40

9.2

Processed fish

10

0.3

20

Miscellaneous (toppings only)

10–20

1.8

The intakes predicted in the USA were based on the same maximum levels of use in 11 categories of foods (Table 4) and the consumption of these foods in the USA from a dietary survey in 1994–96 (US Department of Agriculture, 1998). In this 3-year national survey, data were collected from two 24-h recalls from representative households. The predicted dietary intakes by children, adolescents, and adults are shown in Table 5. For adults, the predicted intake of trehalose from all proposed uses except chewing-gum was 7.2 g/day at the mean of consumption and 16 g/day at the 90th percentile. The predicted mean intake by eating occasion (excluding extended eating occasions) ranged from 3.9 to 9.7 g per occasion, while the predicted intake at the 90th percentile ranged from 7.6 to 19 g per occasion. The highest predicted intake from an individual food was due to consumption of ice cream. Intake from chewing-gum was estimated separately, as consumption of this commodity was not included in the survey but was based on a postal survey of 1500 individuals in1995 who reported their 1-day intake of regular and sugar-free chewing-gum. It was assumed that trehalose comprised 10% of the total weight of chewing-gum from its use as a non-hygroscopic sweetener in the coating. The predicted mean intake from use of chewing-gum was 0.4 g/day, and that for the 90th percentile of consumption was 0.8 g/day (Murphy & Kruger, 2000).

Table 4. Food uses of trehalose and maximum levels of use in the USA

Food category

Concentration of trehalose (%)

Maximum level of use (%)

Bakery cream

20–25

5–6

Confectionery

7

Cookies

10

Hard candies

20

Ice cream

10

Icings

25

5

Instant noodles/rice

2

Processed fruit
(jams, fillings, toppings)

10–20

Restructured sea-food

10

Sponge cake

8–10

Sugar coatings

50

10

Table 5. Predicted intake of trehalose from all proposed uses in food in populations in the USA

Age group (years)

Eating occasion

No. of occasions of use

Intake per user (g)

Mean

90th percentile

Children (2–12)

Per day

3251

5.2

11

Breakfast

3.6

5.8

Lunch

1072

4.1

7.8

Dinner

482

4.8

9.2

Snack

2038

3.7

7.5

Other

53

5.8

12

Adolescents (13–19)

Per day

866

7.5

15

Breakfast

221

5.1

10

Lunch

207

6.6

12

Dinner

122

7.1

17

Snack

589

5.2

9.9

Other

17

5.9

10

Adults (

ł 20)

Per day

6384

7.2

16

Breakfast

2142

3.9

7.6

Lunch

1277

6.1

15

Dinner

1485

8.1

19

Snack

3084

5.5

12

Other

121

9.7

16

The predicted mean intake of of trehalose by Australian consumers thus ranged from 6.5 to 11 g/day; the predicted daily intake (including chewing-gum) by adult consumers at the 90th percentile in the USA was < 17 g/day, while the intake per occasion at the 90th percentile could reach 19 g. The Commmittee noted that the predictive models give overestimates of trehalose intake because they assume that all foods in named use categories contain trehalose at maximum levels. In addition, the predicted intakes of high consumers are overestimated by use of 24-h dietary recall records in the food consumption surveys in both Australia and the USA, which tend to overestimate habitual intakes of foods (Institute of European Studies, 1998).

4. COMMENTS

Trehalose is hydrolysed to glucose by the enzyme trehalase, which is located in the intestinal mucosa, and the small amount of intact trehalose that may be absorbed is metabolized by trehalase in the blood plasma, the liver, or the kidney. Trehalase deficiency has been identified in some individuals, but its prevalence appears to be very low in most populations, with the possible exception of that of Greenland, where an 8% prevalence has been reported.

Studies in which trehalose was administered in the diet have been performed in mice and dogs. In a 3-month study in mice, slight, sporadic changes in clinical biochemistry were seen in males at the highest dose tested, 7.3 g/kg bw per day, but there was no evidence of pathological alterations. In a 14-day study in dogs, no clinical or morphological evidence of toxicity was seen at 5 g/kg bw per day, which was the highest dose tested.

In a two-generation study in rats, no effect was found on reproduction. Similarly, in studies of developmental toxicity in rats and rabbits, there was no evidence of teratogenicity. The results of assays for genotoxicity were negative. No long-term studies were available, but these were considered unnecessary since trehalose is rapidly metabolized to glucose at the levels of intake predicted from the proposed uses.

The toxicological data available on the enzymes used in the preparation of trehalose, some of which have been evaluated by the Committee previously, did not raise any concern.

Studies in humans indicate that trehalose is well tolerated. Increased frequencies of malabsorption and gastrointestinal symptoms were noted in individuals consuming single doses of 20 g or more. In the limited data on individuals with known or suspected trehalase deficiency, the only effects seen were the gastrointestinal effects expected of an undigested disaccharide.

The daily intake of trehalose was predicted on the basis of conservative assumptions, by combining the highest proposed levels of use. For adults in the USA, the mean predicted intake from all proposed uses, except chewing-gum, was 7 g/day, and that of consumers at the 90th percentile was 16 g/day. The mean intake per eating occasion ranged from 4 to 10 g, while intake of consumers at the 90th percentile ranged from 8 to 19 g per occasion. Average users of chewing-gum and those at the 90th percentile of use would ingest 0.4 and 0.8 g/day of trehalose, respectively. For Australian adults, the predicted mean intake of trehalose (including chewing-gum) ranged from 6 to 10 g/day. However, the data from both Australia and the USA were based on short-term dietary recall, which tends to result in overestimates of habitual intake.

5. EVALUATION

On the basis of the available information, the Committee established an ADI for trehalose ‘not specified’2.

Institute of European Studies (1998) The Effect of Survey Duration on the Estimation of Food Chemical Intakes, IEFS Report No 3.

Kitching, J. (1995) Trehalose (crystal) bacterial mutation assay. Unpublished report from Huntingdon Research Centre Ltd, Huntingdon, United Kingdom. Submitted to WHO by the Flavor and Extract Manufacturers’ Association of the United States.

Lina, B.A.R. (1999) Sub-chronic (13-wk) oral toxicity study with isomylase in rats. Unpublished report No. V99.646 from TNO Nutrition and Food Research Institute, Zeist, Netherlands. Submitted to WHO by the Flavor and Extract Manufacturers Association of the United States.

Murphy, M.M. & Kruger, C.L. (2000) Estimated intake of trehalose in the US based upon proposed use levels in 11 categories of foods. Unpublished report from Environ Corp. Submitted to WHO by Bioresco Inc., Switzerland.

ADI "not specified" is used to refer to a food substance of very low toxicity which, on the basis of the available data (chemical, biochemical, toxicological and other) and the total dietary intake of the substance arising from its use at the levels necessary to achieve the desired effect and from its acceptable background levels in food, does not, in the opinion of the Committee, represent a hazard to health. For that reason, and for reasons stated in the individual evaluation, the establishment of an ADI expressed in numerical form is not deemed necessary. An additive that meets this criterion must be used within the bounds of good manufacturing practice, i.e. it should be technologically efficacious and should be used at the lowest level necessary to achieve this effect, it should not conceal food of inferior quality or adulturated food, and it should not create a nutritional imbalance.

2

ADI "not specified" is used to refer to a food substance of very low toxicity which, on the basis of the available data (chemical, biochemical, toxicological and other) and the total dietary intake of the substance arising from its use at the levels necessary to achieve the desired effect and from its acceptable background levels in food, does not, in the opinion of the Committee, represent a hazard to health. For that reason, and for reasons stated in the individual evaluation, the establishment of an ADI expressed in numerical form is not deemed necessary. An additive that meets this criterion must be used within the bounds of good manufacturing practice, i.e. it should be technologically efficacious and should be used at the lowest level necessary to achieve this effect, it should not conceal food of inferior quality or adulturated food, and it should not create a nutritional imbalance.